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The role of tinman, a mesodermal cell fate gene, in axon pathfinding during the development of the transverse nerve in Drosophila.

During the development of peripheral nerves, pioneer axons often navigate over mesodermal tissues. In this paper, we examine the role of the mesodermal cell determination gene tinman on cells that provide pathfinding cues in Drosophila. We focus on a subset of peripheral nerves, the transverse nerves, that innervate abdominal segments. During wildtype embryonic development, the transverse nerve efferents associate with glial cells located on the dorsal aspect of the CNS midline (transverse nerve exit glia). These glial cells have cytoplasmic extensions that prefigure the transverse nerve pathway from the CNS to the body wall musculature prior to transverse nerve formation. Transverse nerve efferents extend along this scaffold to the periphery, where they fasciculate with projections from a peripheral neuron--the LBD. In tinman mutants, the transverse nerve exit glia appear to be missing, and efferent fibers remain stalled at the CNS midline, without forming transverse nerves. In addition, fibers of the LBD neurons are often truncated. These results suggest that the lack of exit glia prevents normal transverse nerve pathfinding. Another prominent defect in tinman is the loss of all dorsal neurohemal organs, FMRFamide-expressing thoracic structures which likely contain the homologs of the transverse nerve exit glia in the thoracic segments. Our results support the hypothesis that the exit glia have a mesodermal origin and that glia play an essential role in determining transverse nerve axon pathways.

Animals↗

Role of MAP kinase in mesoderm induction and axial patterning during Xenopus development.

We have examined the role of MAP kinase during mesoderm induction and axial patterning in Xenopus embryos. MAP Kinase Phosphatase (MKP-1) was used to inactivate endogenous MAP kinase and was found to prevent the induction of early and late mesodermal markers by both FGF and activin. In whole embryos, MKP-1 was found to disrupt posterior axial patterning, generating a phenotype similar to that obtained with a dominant inhibitory FGF receptor. Overexpression of either constitutively active MAP kinase or constitutively active MAP kinase (MEK) was sufficient to induce Xbra expression, while only constitutively active MEK was able to significantly induce expression of muscle actin. When MAP kinase phosphorylation was used as a sensitive marker of FGF receptor activity in vivo, this activity was found to persist at a low and relatively uniform level throughout blastula stage embryos. The finding that a low level of MAP kinase phosphorylation exists in unstimulated animal caps and is absent in caps overexpressing a dominant inhibitory FGF receptor provides a basis for our previous observation that overexpression of this receptor inhibits activin induction. These results indicate that FGF-dependent MAP kinase activity plays a critical role in establishing the responsiveness of embryonic tissues to mesoderm inducers.

Activins↗

Clustered arrangement of winged helix genes fkh-6 and MFH-1: possible implications for mesoderm development.

The 'winged helix' or 'forkhead' transcription factor gene family is defined by a common 100 amino acid DNA binding domain which is a variant of the helix-turn-helix motif. Here we describe the structure and expression of the mouse fkh-6 and MFH-1 genes. Both genes are expressed in embryonic mesoderm from the headfold stage onward. Transcripts for both genes are localised mainly to mesenchymal tissues, fkh-6 mRNA is enriched in the mesenchyme of the gut, lung, tongue and head, whereas MFH-1 is expressed in somitic mesoderm, in the endocardium and blood vessels as well as the condensing mesenchyme of the bones and kidney and in head mesenchyme. Both genes are located within a 10 kb region (in mouse chromosome 8 at 5.26 +/- 2.56 cM telomeric to Actsk1. The close physical linkage of these two winged helix genes is conserved in man, where the two genes map to chromosome 16q22-24. This tandem arrangement suggests the common use of regulatory mechanisms. The fkh-6/MFH-1 locus maps close to the mouse mutation amputated, which is characterised by abnormal development of somitic and facial mesoderm. Based on the expression patterns we suggest that a mutation in MFH-1, not fkh-6 is the possible cause for the amputated phenotype.

Amino Acid Sequence↗

Expression of Pax-3 is initiated in the early neural plate by posteriorizing signals produced by the organizer and by posterior non-axial mesoderm.

Pax-3 is a paired-type homeobox gene that is specifically expressed in the dorsal and posterior neural tube. We have investigated inductive interactions that initiate Pax-3 transcript expression in the early neural plate. We present several lines of evidence that support a model where Pax-3 expression is initiated by signals that posteriorize the neuraxis, and then secondarily restricted dorsally in response to dorsal-ventral patterning signals. First, in chick and Xenopus gastrulae the onset of Pax-3 expression occurs in regions fated to become posterior CNS. Second, Hensen's node and posterior non-axial mesoderm which underlies the neural plate induce Pax-3 expression when combined with presumptive anterior neural plate explants. In contrast, presumptive anterior neural plate explants are not competent to express Pax-3 in response to dorsalizing signals from epidermal-ectoderm. Third, in a heterospecies explant recombinant assay with Xenopus animal caps (ectoderm) as a responding tissue, late, but not early, Hensen's node induces Pax-3 expression. Chick posterior non-axial mesoderm also induces Pax-3, provided that the animal caps are neuralized by treatment with noggin. Finally we show that the putative posteriorizing factors, retinoic acid and bFGF, induce Pax-3 in neuralized animal caps. However, blocking experiments with a dominant-inhibitory FGF receptor and a dominant-inhibitory retinoic acid receptor suggest that Pax-3 inductive activities arising from Hensen's node and posterior non-axial mesoderm do not strictly depend on FGF or retinoic acid.

Animals↗

Induction of identified mesodermal cells by CNS midline progenitors in Drosophila.

The Drosophila ventral midline cells generate a discrete set of CNS lineages, required for proper patterning of the ventral ectoderm. Here we provide the first evidence that the CNS midline cells also exert inductive effects on the mesoderm. Mesodermal progenitors adjacent to the midline progenitor cells give rise to ventral somatic mucles and a pair of unique cells that come to lie dorsomedially on top of the ventral nerve cord, the so-called DM cells. Cell ablation as well as cell transplantation experiments indicate that formation of the DM cells is induced by midline progenitors in the early embryo. These results are corroborated by genetic analyses. Mutant single minded embryos lack the CNS midline as well as the DM cells. Embryos mutant for any of the spitz group genes, which primarily express defects in the midline glial cell lineages, show reduced formation of the DM cells. Conversely, directed overexpression of secreted SPITZ by some or all CNS midline cells leads to the formation of additional DM cells. Furthermore we show that DM cell development does not depend on the absolute concentration of a local inductor but appears to require a graded source of an inducing signal. Thus, the Drosophila CNS midline cells play a central inductive role in patterning the mesoderm as well as the underlying ectoderm.

Animals↗

Control of cell fates and segmentation in the Drosophila mesoderm.

The primordia for heart, fat body, and visceral and somatic muscles arise in specific areas of each segment in the Drosophila mesoderm. We show that the primordium of the somatic muscles, which expresses high levels of twist, a crucial factor of somatic muscle determination, is lost in sloppy-paired mutants. Simultaneously, the primordium of the visceral muscles is expanded. The visceral muscle and fat body primordia require even-skipped for their development and the mesoderm is thought to be unsegmented in even-skipped mutants. However, we find that even-skipped mutants retain the segmental modulation of the expression of twist. Both the domain of even-skipped function and the level of twist expression are regulated by sloppy-paired. sloppy-paired thus controls segmental allocation of mesodermal cells to different fates.

Animals↗

Two novel chick T-box genes related to mouse Brachyury are expressed in different, non-overlapping mesodermal domains during gastrulation.

The mouse Brachyury (T) gene plays critical roles in the genesis of normal mesoderm during gastrulation and in the maintenance of a functioning notochord. Abrogation of Brachyury (T) expression within the chordamesoderm of homozygous null mutants nevertheless spares anterior axis formation. An intriguing possibility to explain the preservation of anterior axis formation in these mutants would be the existence of other genes compensating for the loss of Brachyury. This compensation and the recent demonstration that Brachyury is the prototype for an evolutionarily conserved family, prompted a search for other T-box genes participating in axis formation. The chick Brachyury orthologue and two related chick T-box genes that are expressed at the onset of gastrulation have been isolated. One of these novel genes (Ch-TbxT) becomes restricted to the axial mesoderm lineage and is a potential candidate for complementing or extending Brachyury function in the anterior axis (formation of the head process, prechordal plate). The other gene (Ch-Tbx6L), together with chick T, appears to mark primitive streak progenitors before gastrulation. As cells leave the primitive streak, Ch-Tbx6L becomes restricted to the early paraxial mesoderm lineage and could play a role in regulating somitogenesis.

Amino Acid Sequence↗

A Xenopus type I activin receptor mediates mesodermal but not neural specification during embryogenesis.

Activins and other ligands in the TGFbeta superfamily signal through a heteromeric complex of receptors. Disruption of signaling by a truncated type II activin receptor, XActRIIB (previously called XAR1), blocks mesoderm induction and promotes neuralization in Xenopus embryos. We report the cloning and characterization of a type I activin receptor, XALK4. Like truncated XActRIIB, a truncated mutant (tXALK4) blocks mesoderm formation both in vitro and in vivo; moreover, an active form of the receptor induces mesoderm in a ligand-independent manner. Unlike truncated XActRIIB, however, tXALK4 does not induce neural tissue. This difference is explained by the finding that tXALK4 does not block BMP4-mediated epidermal specification, while truncated XActRIIB inhibits all BMP4 responses in embryonic explants. Thus, the type I and type II activin receptors are involved in overlapping but distinct sets of embryonic signaling events.

Activin Receptors↗

A role for the vegetally expressed Xenopus gene Mix.1 in endoderm formation and in the restriction of mesoderm to the marginal zone.

We have studied the role of the activin immediate-early response gene Mix.1 in mesoderm and endoderm formation. In early gastrulae, Mix.1 is expressed throughout the vegetal hemisphere, including marginal-zone cells expressing the trunk mesodermal marker Xbra. During gastrulation, the expression domains of Xbra and Mix.1 become progressively exclusive as a result of the establishment of a negative regulatory loop between these two genes. This mutual repression is important for the specification of the embryonic body plan as ectopic expression of Mix.1 in the Xbra domain suppresses mesoderm differentiation. The same effect was obtained by overexpressing VP16Mix.1, a fusion protein comprising the strong activator domain of viral VP16 and the homeodomain of Mix.1, suggesting that Mix.1 acts as a transcriptional activator. Mix.1 also has a role in endoderm formation. It cooperates with the dorsal vegetal homeobox gene Siamois to activate the endodermal markers edd, Xlhbox8 and cerberus in animal caps. Conversely, vegetal overexpression of enRMix.1, an antimorphic Mix.1 mutant, leads to a loss of endoderm differentiation. Finally, by targeting enRMix.1 expression to the anterior endoderm, we could test the role of this tissue during embryogenesis and show that it is required for head formation.

Animals↗

MyoD and the specification of muscle and non-muscle fates during postembryonic development of the C. elegans mesoderm.

Basic-helix-loop helix factors of the myoD/myf5/ myogenin/MRF4 family have been implicated in acquisition and elaboration of muscle cell fates. Here we describe both myogenic and non-myogenic roles for the Caenorhabditis elegans member of this family (CeMyoD) in postembryonic mesodermal patterning. The postembryonic mesodermal lineage in C. elegans provides a paradigm for many of the issues in mesodermal fate specification: a single mesoblast ('M') divides to generate 14 striated muscles, 16 non-striated muscles, and two non-muscle cells. To study CeMyoD function in the M lineage, we needed to circumvent an embryonic requirement for the protein. Two approaches were used: (1) isolation of mutants that decrease CeMyoD levels while retaining viability, and (2) analysis of genetic mosaics that had lost CeMyoD in the M lineage. With either manipulation, we observed a series of cell-fate transformations affecting a subset of both striated muscles and non-muscle cells. In place of these normal fates, the affected lineages produced a number of myoblast-like cells that initially failed to differentiate, instead swelling to acquire a resemblance to sex myoblasts (M-lineage-derived precursors to non-striated uterine and vulval muscles). Like normal sex myoblasts, the ectopic myoblast-like cells were capable of migration and proliferation followed by differentiation of progeny cells into vulval and uterine muscle. Our results demonstrate a cell-intrinsic contribution of CeMyoD to specification of both non-muscle and muscle fates.

Animals↗

SCL specifies hematopoietic mesoderm in Xenopus embryos.

Targeted gene disruption experiments in the mouse have demonstrated an absolute requirement for several transcription factors for the development of hematopoietic progenitors during embryogenesis. Disruption of the basic helix-loop-helix gene SCL (stem cell leukemia) causes a block early in the hematopoietic program with defects in all hematopoietic lineages. To understand how SCL participates in the organogenesis of blood, we have isolated cDNAs encoding Xenopus SCL and characterized the function of SCL during embryogenesis. We demonstrate that SCL is expressed in ventral mesoderm early in embryogenesis. SCL expression is induced by BMP-4, and a dominant negative BMP-4 receptor inhibits SCL expression in the ventral region of the embryo. Expression of SCL in either bFGF-treated animal pole explants or dorsal marginal zone explants leads to the expression of globin protein. Furthermore, over-expression of SCL does not alter normal dorsal-ventral patterning in the embryo, indicating that SCL acts to specify mesoderm to a hematopoietic fate after inductive and patterning events have occurred. We propose that SCL is both necessary and sufficient to specify hematopoietic mesoderm, and that it has a similar role in specifying hematopoietic cell fate as MyoD has in specifying muscle cell fate.

Amino Acid Sequence↗

Requirement for Xvent-1 and Xvent-2 gene function in dorsoventral patterning of Xenopus mesoderm.

Xvent-1 and Xvent-2 are members of a novel homeobox subfamily that have been implicated in dorsoventral patterning in Xenopus mesoderm and are thought to function in BMP signalling. Here we investigate the requirement for Xvent function by employing two dominant-negative strategies. Loss of Xvent function dorsalizes ventral mesoderm, induces secondary embryonic axes and directly neuralizes ectoderm. We further find that (1) Xvents act as transcriptional repressors, (2) Xvents function in an additive fashion and (3) a surprising number of genes are able to rescue dominant-negative Xvent phenotypes including Bmp-4, Smad-1 and wild-type Xvents and Xhox3, but not Xwnt-8. The results show that Xvent-1 and Xvent-2 are essential for ventral mesoderm formation and for preventing neural differentiation. A model is suggested to explain how Bmp-4 positional information is converted into distinct cellular responses.

Animals↗

Induction of the mesendoderm in the zebrafish germ ring by yolk cell-derived TGF-beta family signals and discrimination of mesoderm and endoderm by FGF.

The endoderm forms the gut and associated organs, and develops from a layer of cells which emerges during gastrula stages in the vertebrate embryo. In comparison to mesoderm and ectoderm, little is known about the signals which induce the endoderm. The origin of the endoderm is intimately linked with that of mesoderm, both by their position in the embryo, and by the molecules that can induce them. We characterised a gene, zebrafish gata5, which is expressed in the endoderm from blastula stages and show that its transcription is induced by signals originating from the yolk cell. These signals also induce the mesoderm-expressed transcription factor no tail (ntl), whose initial expression coincides with gata5 in the cells closest to the blastoderm margin, then spreads to encompass the germ ring. We have characterised the induction of these genes and show that ectopic expression of activin induces gata5 and ntl in a pattern which mimics the endogenous expression, while expression of a dominant negative activin receptor abolishes ntl and gata5 expression. Injection of RNA encoding a constitutively active activin receptor leads to ectopic expression of gata5 and ntl. gata5 is activated cell-autonomously, whereas ntl is induced in cells distant from those which have received the RNA, showing that although expression of both genes is induced by a TGF-beta signal, expression of ntl then spreads by a relay mechanism. Expression of a fibroblast growth factor (eFGF) or a dominant negatively acting FGF receptor shows that ntl but not gata5 is regulated by FGF signalling, implying that this may be the relay signal leading to the spread of ntl expression. In embryos lacking both squint and cyclops, members of the nodal group of TGF-beta related molecules, gata5 expression in the blastoderm is abolished, making these factors primary candidates for the endogenous TGF-beta signal inducing gata5.

Activins↗

Vegetal rotation, a new gastrulation movement involved in the internalization of the mesoderm and endoderm in Xenopus.

A main achievement of gastrulation is the movement of the endoderm and mesoderm from the surface of the embryo to the interior. Despite its fundamental importance, this internalization process is not well understood in amphibians. We show that in Xenopus, an active distortion of the vegetal cell mass, vegetal rotation, leads to a dramatic expansion of the blastocoel floor and a concomitant turning around of the marginal zone which constitutes the first and major step of mesoderm involution. This vigorous inward surging of the vegetal region into the blastocoel can be analyzed in explanted slices of the gastrula, and is apparently driven by cell rearrangement. Thus, the prospective endoderm, previously thought to be moved passively, provides the main driving force for the internalization of the mesendoderm during the first half of gastrulation. For further involution, and for normal positioning of the involuted mesoderm and its rapid advance toward the animal pole, fibronectin-independent interaction with the blastocoel roof is required.

Animals↗

SHH-N upregulates Sfrp2 to mediate its competitive interaction with WNT1 and WNT4 in the somitic mesoderm.

Dorsoventral polarity of the somitic mesoderm is established by competitive signals originating from adjacent tissues. The ventrally located notochord provides the ventralizing signals to specify the sclerotome, while the dorsally located surface ectoderm and dorsal neural tube provide the dorsalizing signals to specify the dermomyotome. Noggin and SHH-N have been implicated as the ventralizing signals produced by the notochord. Members of the WNT family of proteins, on the other hand, have been implicated as the dorsalizing signals derived from the ectoderm and dorsal neural tube. When presomitic explants are confronted with cells secreting SHH-N and WNT1 simultaneously, competition to specify the sclerotome and dermomyotome domains within the naive mesoderm can be observed. Here, using these explant cultures, we provide evidence that SHH-N competes with WNT1, not only by upregulating its own receptor Ptc1, but also by upregulating Sfrp2 (Secreted frizzled-related protein 2), which encodes a potential WNT antagonist. Among the four known Sfrps, Sfrp2 is the only member expressed in the sclerotome and upregulated by SHH-N recombinant protein. We further show that SFRP2-expressing cells can reduce the dermomyotome-inducing activity of WNT1 and WNT4, but not that of WNT3a. Together, our results support the model that SHH-N at least in part employs SFRP2 to reduce WNT1/4 activity in the somitic mesoderm.

3T3 Cells↗

A transitional stage in the commitment of mesoderm to hematopoiesis requiring the transcription factor SCL/tal-1.

In this report, we describe the identification and characterization of an early embryoid body-derived colony, termed the transitional colony, which contains cell populations undergoing the commitment of mesoderm to the hematopoietic and endothelial lineages. Analysis of individual transitional colonies indicated that they express Brachyury as well as flk-1, SCL/tal-1, GATA-1, (beta)H1 and (beta)major reflecting the combination of mesodermal, hematopoietic and endothelial populations. This pattern differs from that found in the previously described hemangioblast-derived blast cell colonies in that they typically lacked Brachyury expression, consistent with their post-mesodermal stage of development (Kennedy, M., Firpo, M., Choi, K., Wall, C., Robertson, S., Kabrun, N. and Keller, G. (1997) Nature 386, 488-493). Replating studies demonstrated that transitional colonies contain low numbers of primitive erythroid precursors as well as a subset of precursors associated with early stage definitive hematopoiesis. Blast cell colonies contain higher numbers and a broader spectrum of definitive precursors than found in the transitional colonies. ES cells homozygous null for the SCL/tal-1 gene, a transcription factor known to be essential for development of the primitive and definitive hematopoietic systems, were not able to form blast colonies but did form transitional colonies. Together these findings suggest that the transitional colony represents a stage of development earlier than the blast cell colony and one that uniquely defines the requirement for a functional SCL/tal-1 gene for the progression to hematopoietic commitment.

Animals↗

Overlapping roles of two Hox genes and the exd ortholog ceh-20 in diversification of the C. elegans postembryonic mesoderm.

Members of the Hox family of homeoproteins and their cofactors play a central role in pattern formation of all germ layers. During postembryonic development of C. elegans, non-gonadal mesoderm arises from a single mesoblast cell M. Starting in the first larval stage, M divides to produce 14 striated muscles, 16 non-striated muscles, and two non-muscle cells (coelomocytes). We investigated the role of the C. elegans Hox cluster and of the exd ortholog ceh-20 in patterning of the postembryonic mesoderm. By examining the M lineage and its differentiation products in different Hox mutant combinations, we found an essential but overlapping role for two of the Hox cluster genes, lin-39 and mab-5, in diversification of the postembryonic mesoderm. This role of the two Hox gene products required the CEH-20 cofactor. One target of these two Hox genes is the C. elegans twist ortholog hlh-8. Using both in vitro and in vivo assays, we demonstrated that twist is a direct target of Hox activation. We present evidence from mutant phenotypes that twist is not the only target for Hox genes in the M lineage: in particular we show that lin-39 mab-5 double mutants exhibit a more severe M lineage defect than the hlh-8 null mutant.

Animals↗

The lefty-related factor Xatv acts as a feedback inhibitor of nodal signaling in mesoderm induction and L-R axis development in xenopus.

In mouse, lefty genes play critical roles in the left-right (L-R) axis determination pathway. Here, we characterize the Xenopus lefty-related factor antivin (Xatv). Xatv expression is first observed in the marginal zone early during gastrulation, later becoming restricted to axial tissues. During tailbud stages, axial expression resolves to the neural tube floorplate, hypochord, and (transiently) the notochord anlage, and is joined by dynamic expression in the left lateral plate mesoderm (LPM) and left dorsal endoderm. An emerging paradigm in embryonic patterning is that secreted antagonists regulate the activity of intercellular signaling factors, thereby modulating cell fate specification. Xatv expression is rapidly induced by dorsoanterior-type mesoderm inducers such as activin or Xnr2. Xatv is not an inducer itself, but antagonizes both Xnr2 and activin. Together with its expression pattern, this suggests that Xatv functions during gastrulation in a negative feedback loop with Xnrs to affect the amount and/or character of mesoderm induced. Our data also provide insights into the way that lefty/nodal signals interact in the initiation of differential L-R morphogenesis. Right-sided misexpression of Xnr1 (endogenously expressed in the left LPM) induces bilateral Xatv expression. Left-sided Xatv overexpression suppresses Xnr1/XPitx2 expression in the left LPM, and leads to severely disturbed visceral asymmetry, suggesting that active 'left' signals are critical for L-R axis determination in frog embryos. We propose that the induction of lefty/Xatv in the left LPM by nodal/Xnr1 provides an efficient self-regulating mechanism to downregulate nodal/Xnr1 expression and ensure a transient 'left' signal within the embryo.

Amino Acid Sequence↗